1. Field of the Invention
The present invention relates to a variation detection method and a system for it useful for screening of a gene variation etc.
2. Related Background Art
One of the techniques for sequencing nucleic acid etc. or for detecting the sequence is to utilize a DNA array. U.S. Pat. No. 5,445,934 discloses a DNA array where 100,000 or more oligonucleotide probes are bonded in 1 inch square. Such a DNA array has an advantage that many characteristics can be examined at the same time with a very small sample amount. When a fluorescence-labeled sample is poured onto such a DNA chip, DNA fragments in the sample bind to probes having a complementary sequence fixed on the DNA chip, and only that part can be discriminated by fluorescence to elucidate the sequence of the DNA fragment in the DNA sample.
Sequencing By Hybridization (SBH) is a method for examining the base sequence utilizing such a DNA array and the details are described in U.S. Pat. No. 5,202,231. In the SBH method, all possible sequences of an oligonucleotide of a certain length are arranged on the substrate, then fully matched hybrids formed by hybridization reaction between probes and the sample DNA are detected. If a set of fully matched hybrids is obtained, the set will give an assembly of overlapping sequences with one base shift being a part of one certain sequence, which sequence is extracted for calling.
Including the SBH method, when complementariness between an oligonucleotide and a sample DNA is examined, it is very difficult to call whether a hybrid was formed or not using one probe for one test item, since the stability of a hybrid differs sequence to sequence, and there is no perfect signal for calling the full complementariness. Science vol. 274 p. 610-614, 1996 discloses a method for calling by comparing the signal intensity of a perfect match hybrid and the weaker intensities of one-base mismatch hybrids. In this method, 15-mer oligonucleotide probes, differing from each other only by one mismatching base at the center of the sequence, are prepared, and the fluorescence intensities of the hybrids of the probes are compared. When the intensity of the full matched hybrid is stronger than that of other hybrids by a predetermined rate, it is called positive.
Further, U.S. Pat. No. 5,733,729 discloses a method using a computer to differentiate a base sequence of a sample from a comparison of fluorescence intensities of obtained hybrids for more accurate calling.
However, the actual binding strength of a hybrid depends on the GC content etc., and difference of the fluorescence intensity between a full match hybrid and a one-base mismatch hybrid also varies in a considerable range depending on the sequence. Thus, a method for calling whether a sequence is fully complementary to a probe or not, using a 15 mer oligonucleotide probe to compare it with other three probes having one mismatched base at the center thereof, can provide more accuracy if each stability is evaluated theoretically or empirically before comparison.
In addition, accurate calling requires precise quantification of signals, and therefore, precision apparatuses such as a confocal laser microscope. Furthermore, in order to measure the fluorescence intensity of a hybrid of every probe and to determine the gene sequence by analyzing the data, a large-scale computer apparatus as well as a detection apparatus for reading the arrays are further required. Therefore, this is a big obstacle for ready use of the DNA array.
On the other hand, gene diagnosis using such a DNA array may be used in group medical examination, individual gene examination or gene-polymorphism study. In such a case, however, the above described precise measurement and analysis are not always required, where a large amount of samples are rapidly treated at a low cost in order to find out variated samples concerning a specific item from a large number of normal samples. Further, the precision apparatus and analysis as described above will be expensive. Accordingly, a concept that screening of the presence or absence of a variation is first performed, and then, detailed examinations of the samples suspected of variation are carried out by screening, saving both time and cost.